Vol. 10(52), pp. 4762-4769, 24 December, 2015 DOI: 10.5897/AJAR2015.9995 Article Number: 18B8E0456476 African Journal of Agricultural ISSN 1991-637X Copyright ©2015 Research Author(s) retain the copyright of this article http://www.academicjournals.org/AJAR

Full Length Research Paper

Possibility of using the intermediate mature stage of kola heckel seeds to shorten the germination time

Edmond Kouablan KOFFI1*, Anatole Kanga N’GUESSAN1, Christophe N’guessan KOUAME1, Modeste Kan KOUASSI1 and Jane Wanjiku KAHIA2

1Centre National de Recherche Agronomique (CNRA) Côte d’Ivoire, Laboratoire Central de Biotechnologies (LCB), KM 17 Route de Dabou, 01 BP 1740 Abidjan, Côte d’Ivoire. 2World Agroforestry Centre (ICRAF) Cote d’ Ivoire Country Program Cocody Mermoz, Abidjan, Côte d’Ivoire.

Received 9 June, 2015; Accepted 25 September, 2015

Garcinia kola is used as chew-stick for dental care in West Africa. The species is nearly commercially extinct in nature, and its propagation is hampered by seed germination problems. The current study investigated seed germination responses of Garcinia kola at different maturity stage. The experiments were conducted at the Central Laboratory of Biotechnology of the Centre National de Recherche Agronomique (CNRA) located at Adiopodoumé (North Abidjan - Côte d’Ivoire) while the fruit samples were collected from Yapo a protected area with very humid equatorial climate. The experimental design was factorial design with two factors. Seeds processed from green fruits of 6 weeks old did not germinate, whilst those of 10 weeks old fruits, at the intermediate stage, germinated at the rate of 46%. Seeds from orange-coloured mature fruits had a germination rate of 54 to 62%. The adventitious roots of intermediate maturity seeds protruded at the same time as mature ones after 9 weeks of sowing and normal germination, with first shoot growth occurring simultaneously after 12 weeks in the two (intermediate maturity and full maturity) stage of fruits. Desiccation tests after 4 weeks revealed similar trends of weight decrease and moisture content in the two kinds of seed. Seeds from partly intermediate mature fruits showed germinating characteristics comparable to mature fruits. These indicate that the seeds from this stage could be used to germinate Garcinia kola Heckel, for conservation, post harvesting and commercial purposes.

Key words: Garcinia kola, planting material, germination, seeds, fruits, Côte d’Ivoire.

INTRODUCTION

Taxonomic description of Garcinia kola Unranked: Unranked: Kingdom: Plantae Order: Unranked: Angiosperms Family:

*Corresponding author. E-mail: [email protected].

Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Koffi et al. 4763

Genus: Garcinia scarcity of the population of G. kola in natural . G. Species: G. kola kola is found in a spontaneous status in natural forest. To prevent its disappearance, it is preserved in forest G. kola Heckel belongs to the Guttiferrae (Clusiaceae) reserves or domesticated. Even in the forest reserves, G. family. There are more than 600 species in this genus kola is still endangered as people enter the forest to distributed widely in tropical and subtropical regions and collect fruits by cutting the trees. only 16 species are found in West Africa (Steentoft, The flowering that takes place in December to March 1988). G. kola occurs in the wet and moist semi- leads to production of immature green fruits between May deciduous forest zones. G. kola is a spreading forest tree to June. The second development phase occur in August growing up to 90 feet high and having a girth of over 5 to September with the formation of intermediate mature feet. Flowers appear from December to March or from fruit with partly orange colour and finally the last phase May to August. Male and female flowers are separate that lead to mature fruit with totally orange fruits occur in and distinct. Fruits are smooth, reddish yellow and the October to November. These three maturity stages are pulp orange yellow with three or four seeds. The acid, not strictly separated, so that the maturity stages overlap red, aril-like fruit pulp is edible. The twigs can be used as from one to another. Seed development is in accordance tapers and the roots yield the favourite bitter chew sticks with fructification stages that are expressed by colours. In sold in small bundles in local markets (Adu-Tutu et al., fact, when fruits are green, seeds are white, not well 1979). The seeds are an important product in West Africa developed (smooth) and are difficult to be extracted from markets. When chewed they have a bitter, astringent the pulp. At the partly orange stage, seed are extractible taste rather resembling that of the raw coffee bean, and from the pulp with a well-developed seed coat and a followed by a slight sweetness. They are chewed as consistent (hard) body. In the last stage where fruits are adjuvant rather than as substitute for the true cola. The totally orange, seeds have the same consistency as in juice has a stimulating effect. Moreover, they also the previous stage with a more distinguishable seed coat enhance the flavour of local alcoholic beverages (Irvine, (Agyili et al., 2007). 1961). As aforementioned, the fruiting phenomenon takes Extracts of various parts of the are used for the place over many months. Conventionally, it is required to treatment of laryngitis, mouth infections, cough, heart wait till the stage of ripe fruits to obtain seedlings or more burn, liver disorder, chest colds hoarseness and others time due to the fact that the degeneration of flesh inflammatory diseases (Ainslie, 1937; Iwu, 1982). The surrounding seeds (pulp) before germination occurs, seeds are also used in the treatment of bronchitis, throat could take one to two months. Due to the long periods it troubles, post-partum haemorrhage, urinary tract takes for the seeds to germinate, it’s of paramount infections and emesis (Irvine, 1961). Complex mixture of importance to evaluate ways to shorten time for the phenolic compounds including biflavonoids, xanthones, seeds to germinate. benzophenones and related triterpenes has been Most of species of the genus Garcinia are sensitive to reported as constituents of this genus (Iwu et al., 1990; desiccation, exhibit low viability and short-lived, and are Geiger and Quine, 1988; Waterman and Hussain, 1983). described as being recalcitrant (Morton, 1987; Chacko The antimicrobial activity of this plant is attributed to the and Pillai, 1997; Geeta et al., 2006). Studies conducted benzophenones and flavanones (Hussain et al., 1982; on G. kola indicated that this specie reduced its viability Iwu et al., 1987). It is also known to have anti-viral, anti- to about 10% at approximately 25 to 27% in moisture inflammatory, anti-diabetic, bronchio-dilator and anti- content categorizing it as recalcitrant in storage heptotoxic attributes. Fruits extracts from G. kola have behaviour (Asomaning et al., 2011). proven effective at stopping the deadly Ebola virus The available literature gives contradictory information replication in laboratory tests (Wikipedia Contributors, concerning the germination of G. kola seeds. Some 2006). The sap of G. kola is used for the treatment of authors (Okafor, 1982; Kengue and Ndo, 2003; Mbolo, parasitic skin diseases while the latex is orally ingested 2002) describe G. kola seed as easy to germinate, while for the treatment of gonorrhoea. It is also useful in the others (Gyimah, 2000; Adebisi, 2004; Anegbeh et al., eradication of guinea worm infestation (Ofakansi et al., 2006) describe it as exhibiting a high degree of 2008). dormancy. According to observations made in Cote The fruits of G. kola in Cote d Ivoire are sold in local d’Ivoire, germination in G. kola could start at least three markets or exported to Sahelian countries where the to four months in ideal conditions and last ten to eleven fruits are in high demand. The export of fruits permits the months after sowing (Agyili et al., 2007). Such duration development of commercial activities that has increased seems obviously relatively long compared to most tree the household incomes of many families. To face the species or non-wood forest products. In such conditions, increasing demand, drastic methods are used to collect reducing germination duration seems quite crucial as G. the fruits. In most of the cases, trees are cut down kola tends to be domesticated according to products and instead of climbing up or using long pole to collect fruits. off products of economic value related to the specie. The harvesting of wood and roots has contributed to the The overall objective of the study was to contribute in 4764 Afr. J. Agric. Res.

A A A B B B C C C

Figure 1. Different stages of maturity in Garcinia kola fruits: (A) green fruits (immature seeds); (B) partly orange (intermediate mature) fruits, and (C) fully orange (mature) fruits.

Fleshy mesocarp

Crossed Encrusted seeds

Figure 2. Cross section of ripe fruit of Garcinia kola showing encrusted seeds patterns in mesocarp and the extracted seeds.

the preservation of G. kola from extinction by surrounding them (Figure 2). Fruits have a thick skin and a pulp that domestication. Specifically, the research conducted must be removed. Removing these parts before extracting seeds aimed at shortening the germination duration process by was found to be very difficult for all the three stages. exploring germination of seeds from different maturity stages during fruiting in order to quicken the process of Experimental procedure producing planting material. Germination tests were conducted in the laboratory and seeds were sown in sand or in sawdust at 1 cm depth with 2 replicates of 25 MATERIALS AND METHODS seeds each. A factorial experimental design comprising two factors was implemented. Factors were the maturity stage of the seeds Sites characteristics (with three levels: immature, intermediate and mature) and the substrate (sawdust and sand). For desiccation tests, two (2) Experiments were conducted at the Central Laboratory of replicates of 65 seeds each were used for the three stages of Biotechnology of the Centre National de Recherche Agronomique maturity. Their weight was followed up for 30 days. Moisture (CNRA) located at Adiopodoumé PK 17 (North Abidjan – Côte content of intermediate and mature seeds was assessed using 50 d’Ivoire). This site is approximately 50 km from the fruit collection seeds according to ISTA (2007). In each phase of maturity, 50 site (Yapo forest). Both sites are located in the southern region of seeds divided into 5 replicates of 10 seeds each were analysed Côte d'Ivoire. Yapo forest is located South-East of Côte d’Ivoire (5° within a period of 4 weeks. 43'13 "N, 4° 9'0" W). It is a protected forest area with equatorial climate, very humid, characterized by long (from April to mid-July) and short (from September to November) rainy seasons with an Data collection average rainfall of 1400 mm separated by dry seasons. The maximum temperature is between 29 and 32°C. Initial characteristics (weight, length, width, water content and . moisture content) of seeds were determined before the germination tests. Seeds from different maturity stages were extracted from Plant material fruits and full seeds with testa were weighed using Sartorius electronic balance. Length and width of seeds were determined Fruits of G. kola were collected from four trees at three different using “Pied à coulisse” instrument. Water content was determined stages of maturity namely immature green fruits of 6 weeks old, according to ISTA (2007). intermediate, partly orange-green fruits of 10 weeks old and from For germination tests, data on the germination rate, the presence mature, fully orange fruits of 14 weeks old (Figure 1). Encrusted or absence of adventitious roots as well as shoots and the time seeds were extracted from fruits by removing the fleshy mesocarp taken for them to appear were recorded. Germination process was

A A A Koffi et al. 4765

Table 1. Weight, length, width and moisture content of Garcinia kola seeds at different stages of maturity.

Parameter Weight (g) Length (cm) Width (cm) Moisture content (%) Immature 2.72  0.69b 3.45  0.34a 2.02  0.21a 65.10  40.37a Intermediate 3.88  1.09b 4.02  0.49a 2.26  0.22a 40.37  2.60b Mature 5.88  0.85a 3.28  0.23a 2.52  0.13a 37.18  2.19b

Values represent means ± SE. Means within a column followed by different letters are significantly different at P = 5%.

Table 2. The effect of sowing substrate on shoots appearance in Garcicina kola mature seeds.

Duration (Week) 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Sawdust 1 0 1 6 0 0 0 10 2 0 1 0 0 0 Substrate Sand 0 0 0 0 0 0 0 0 6 7 15 3 5 1

followed up when seeds were sown. Seeds were removed from occurring during seed maturation could explain such substrates to evaluate germination stage on a weekly basis. The situation whereby maturity is related to water lost and time of root or shoot emergency and the number of seeds seed tissues firmness acquired (Fiona and Roger, 2003). germinated were recorded. Desiccation tests were conducted with seeds from fruits at the When considering the length, intermediate fruits three stages of maturity. Desiccation was carried out in a registered a higher value than both mature and green desiccators whose upper compartment contained seeds while the fruits. Moisture content decrease could be related also to lower compartment was filled with silicagel. The weight of these tissue maturity acquirement. desiccated seeds was measured daily till the weight became stable. Mean weight (of about 5.9 g) and moisture content

(37.2) of mature seeds from Côte d’Ivoire during the Data analysis current study were found to be inferior to that from (Agyili et al., 2007; Asomaning et al., 2011). This result Data (weight, length, width and moisture content) were statistically showed the diversity of seed characteristics that could be analysed for significance by analysis of variance with mean linked to the distribution area of the Garcinia kola species separation by Newman-Keuls test and means were separated using that extend from to Congo (Vivien and a threshold at 5% level of significance with Statistical software (release 7.5). Faure, 1985; Eyog-Matig et al., 2007).

RESULTS AND DISCUSSION Seeds germination tests

Seeds characteristics Seeds from green fruits did not germinate, on sawdust and sand substrates. Germination occurred earlier (9 Seeds weight increased with the maturity. Seeds from weeks) on sawdust compared to 14 week on sand green fruits showed a relative low weight compared to substrate in mature seeds. Seeds sown in sawdust those from intermediate fruits. The later fruits were found germinated faster than those sown in sand (Table 2). The to have lower weight than the mature fruits. It was germination occurred from fully orange fruits after 5 observed that water and moisture content varied with the weeks while they appeared 8 weeks after sowing in the stages of maturation. Mature seeds had a lower water partly orange ones. Mature seeds recorded a relatively and moisture content than that of intermediate fruits. high number of germinated seedlings compared to seeds These seeds recorded lower values compared to from partly orange fruits despite the same evolution of immature seed. The moisture content of intermediate seed germination curves (Figure 3). mature fruits was observed to be close to that one of The primary (false) root appears first (one week) mature fruits but very different compared to immature followed by the root and the shoot (nineth week) (Figure fruits (Table 1). As shown, seeds weight increased 4) which occurred 9 weeks on sawdust and 14 weeks on according to maturation process. However, an opposite sand (Table 2). The mature seeds recorded the highest observation was made with water and moisture content. percentage (56%) of roots appearance, while the In this case, mature seeds expressed lower water and intermediate mature seeds recorded a lower percentage moisture content than that of intermediate ones that of 36%. Concerning the shoot appearance, a rate of 28% indicated lower values of these two characteristics was recorded for the intermediate mature seeds at the compared to non-mature seed. Physiological phenomena 12th week, while at the 13th week mature seeds registered 4766 Afr. J. Agric. Res.

60.00

50.00

40.00

30.00

Number of seedsof Number 20.00

10.00

0.00 Week Week Week Week Week Week Week Week Week Week Week Week Week Week 1-5 6 7 8 9 10 11 12 13 14 15 16 17 18 Duration (Week)

Intermediate stage Mature stage

Figure 3. Germination trend of Garcinia kola seeds (from intermediate mature: partly orange and mature: fully orange fruits) on sawdust. Intermediate stage: mature seeds from partly orange fruits. Mature stage: mature seeds from fully orange fruits.

A B C

A

Figure 4. Germination process steps from mature seeds of Garcinia kola on sawdust: One week (A), 9th week (B) and 13th week (C).

the highest (40%) shoot appearance percentage (Table 13th week in mature seeds and the intermediate mature 3). The highest (40%) number of roots was observed at seeds recorded a lower (24%) number of roots at the Koffi et al. 4767

Table 3. Percentage of shoot and root appearance according to stage of maturity of seeds from Garcinia kola observed over 16 weeks.

Duration (week) Organ Maturity Total 6 8 9 10 12 13 14 16 Intermediate 12 08 36 08 00 00 04 00 68 Roots (%) Mature 16 08 56 04 00 04 04 04 96

Intermediate 00 04 04 00 28 24 08 00 68 Shoot (%) Mature 04 04 24 12 00 40 08 04 96

6.00

5.00

4.00

3.00

Length (cm) Length 2.00

1.00

0.00 Week 6 Week 7 Week 8 Week 9 Week Week Week Week Week Week Week Week Week 10 11 12 13 14 15 16 17 18 Duration (Week)

Interm Stage Mat Stage

Figure 5. Root growth length between seeds from intermediate mature (partly orange) and mature (fully orange) fruits in G. kola. Blue colour: Adventitious root length from intermediate mature seeds from partly orange fruits. Pink colour: Adventitious root length from mature seeds from fully orange fruits. Numbers indicate seeds showing shoots.

same time. It was observed that the mature seeds gave a seeds could be quite advantageous on an economic point lower root length from 6th to 11th week. However, this of view. Water is required for germination as shown with trend was reversed after the 12th week (Figure 5). The intermediate maturity seeds that germinated at the same mature seeds had the highest percentage (96%) time as mature seeds. But the reduced number of germination rate compared to intermediate mature seeds plantlets in intermediate maturity seeds in comparison to (68%) (Table 3). mature seeds could be explained by incomplete Roots and shoot formation appeared at the same physiological maturity of these seeds. Nevertheless, the period in both mature and intermediate mature seeds. germination characteristics in respect to the two However the mature seeds recorded an highest physiological maturity stages could favour the use of germination rate than the intermediate mature seeds. It intermediate maturity seeds. could be assumed that intermediate mature seeds The fact that there was high shoot development in reached the potential maturity needed for germination mature seeds at the 13th week, in comparison to and they could, therefore, be harvested early, in addition intermediate mature seed, could be explained by the to full mature seeds for propagation, postharvest storage, maturity stage (Fiona and Roger, 2003). Related to the conservation and marketing purposes (Ofor et al., 2010). substrate, sawdust used alone or in combination with It takes two months for the intermediate mature seeds to forest soils appeared as a good substrate for seed become mature and thus the use of intermediate mature germination. This result is in agreement with that of 4768 Afr. J. Agric. Res.

450.00 400.00 350.00 300.00 250.00 200.00 150.00

Weight (grammes) Weight 100.00 50.00 0.00 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Duration (Days)

Immature seeds Intermediate seeds Mature seeds

Figure 6. The effect of desiccation of seeds of G. kola on weight. Red curve: Immature seeds from totally green fruits. Green curve: Intermediate mature seeds from partly orange fruits. Blue curve: Mature seeds from fully orange fruits

Eyog-Matig et al. (2007). experimental conditions. The G. kola seeds require higher moisture to germinate compared to orthodox seeds where germination occur at 5% moisture content Desiccation tests after desiccation.

During these tests, it was observed that the weight of seeds decreased and was negatively correlated to the Conclusion stage of maturity. The weight of immature seeds was drastically reduced when compared to that of The exploration of the three stages (Green, partly orange intermediate and mature stage fruits (Figure 6). and totally orange) of maturity of G. kola fruits in The similarity of weight decrease in mature and accordance with seed maturity (immature, intermediate intermediate mature seeds suggests that the two maturity and full maturity) showed that intermediate maturation stages led to a comparable physiological mature seeds had a comparable behaviour with mature maturity. Earlier work on germination tests and seeds in terms of germination. We conclude that, partly desiccation tests were carried out in mature seeds (Agyili orange fruits contain intermediate mature seeds whose et al., 2007; Ofor et al., 2010), while this study, permitted physiological potential is high. These seeds could be a comparative analysis of germination and desiccation harvested to for postharvest storage, conservation and conditions in seeds from partly orange fruits and seeds germination for marketing purposes and propagation of from fully orange fruits. Basically, desiccation tests are planting materials. Such intermediate mature seeds could conducted to confirm whether the seeds are orthodox or allow gain of time to make available sufficient quantity in recalcitrant. In general orthodox seeds are less sensitive the process of plant material production. The mature to desiccation in comparison with recalcitrant ones that seeds of G. kola seeds are known to have numerous do not germinate after removal of water. G. kola seeds medicinal values and we recommend that there is need belongs to the genus Garcinia that is described as having to evaluate the composition of intermediate mature seeds recalcitrant species (Morton, 1987; Chacko and Pillai, using chemical, organic, alkaloid, antioxidant and 1997). Thus, during the current study, the intermediate biochemical tests. mature and mature seed did not germinate after desiccation. These results are contrary to those of Asomaning et al. (2011), who found that the seeds were Conflict of Interests able to germinate up to 30% with low moisture content. This could be probably due to the difference in the The authors have not declared any conflict of interests. Koffi et al. 4769

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